EMBO/SourceData huggingface dataset:
Figure 4B. Mycobactin synthesized by Mtb WT and iniA::hyg grown in low iron conditions. Data are presented as the mean of mycobactin extracted from two independent cultures.C. The Mtb siderophore synthesis mutant (ST142) was cultured in low iron MM (medium) plain or supplemented with MEV preparations derived from equal number of Mtb WT, iniA::hyg and complemented strains grown in low iron MM. ST142 growth was determined based on the number of viable cells detected by the resazurine cell viability assay. MEV obtained from three independent cultures were tested.
Figure 5A. Representative Immunodot-blot of bacterial antigens associated with EVs isolated from the culture supernatants of THP-1 cells infected with Mtb WT, iniA:hyg and complemented strains versus uninfected cells (UI). Anti-MEV specific antiserum was used for detection. The decreasing black bar indicates that increasing dilutions were spotted onto the membrane. The experiment was repeated three times with independent cultures.B. Number of nanoparticles per mL determined by Zeta View NTA in three EV preparations derived from the culture supernatans of THP-1 cells infected with Mtb WT, iniA:hyg and iniA::hyg complemented strains.C. Representative Western blot of total EVs isolated from the CSF of THP-1 cells infected with Mtb WT, iniA::hyg and complemented strains versus uninfected cells (UI) detecting the exosome marker CD9. The experiment was repeated twice with similar results.
Figure 1B HeLa cells expressing the indicated GFP-tagged Nups were semi-permeabilized and incubated with mitotic HeLa cell extract at 30ºC. NPC disassembly was followed by monitoring the fluorescence intensity of the respective Nups at the nuclear rim and influx of TRITC-dextran. Scale bar, 30 μm.C Quantification of dextran-positive nuclei over time of the experiments in (B). N = 3 biological replicates, n > 100 cells. Error bars, SEM.E Quantification of 3GFP-Nup133, GFP-Nup107 and 2GFP-Nup58 intensity at the NE over time of the experiments in (B). Error bars, SEM.
Figure 2B Input, light and dense fractions were analyzed by immunoblotting using the indicated antibodies. The samples were derived from the same experiment but processed using several gels and blots.C Quantification of the percentage of Nups in the light fractions relative to the total amount of Nups over all fractions. N = 4 biological replicates, except for Nup214 (N = 2 biological replicates). Error bars, SEM.
Figure 3B Confocal images of metaphase-arrested HeLa cells expressing 3GFP-Nup133, GFP-Nup107, GFP-Nup53 or 2GFP-Nup58 that were either directly fixed (untreated) or first semi-permeabilized and then fixed. The ER was visualized by immunostaining with an antibody targeting calnexin; DNA was visualized with Hoechst (visible only in the merged channels). Representative images of 3 biological replicates. Scale bar, 10 μm and inset scale bar, 1 μm.
Figure 4A, B 2D dSTORM images of interphase (A) or metaphase (B) U2OS cells expressing SNAP-tagged Nup96. Cells were semi-permeabilized with digitonin and Nup96 was labelled with the BG-AF647 SNAP dye. The SMLM data was reconstructed using Thunderstorm. Left panels were rendered to black and white and enhanced for viewing purposes. Insets (yellow dashed boxes) are displayed to the right. Insets in (A) show ring-shaped structures built by Y-complexes in interphase cells. Insets in (B) represents mitotic clusters outside the spindle area (class 1, inset 1) and at kinetochores (class 2). Scale bar (left panels), 10 μm. Representative images of 4 biological replicates. Inset scale bars, 200 nm.
Figure 5A 2D dSTORM images of a metaphase-arrested U2OS cell expressing Nup96 with a SNAP-tag, labelled with BG-Alexa Fluor 647 (orange), in combination with confocal imaging of the ER immunostained for Climp63 (red) and phospho-histone H3 (cyan) to label DNA. Inset 1 (mitotic clusters) and inset 2 (kinetochores) are indicated with white dashed boxes. SMLM data was reconstructed using Thunderstorm. Representative images of N = 4 biological replicates. Scale bars, 10 μm. Inset scale bars, 200 nm.C Quantification of the number of localizations in interphase NPCs (NPC), mitotic clusters and kinetochores of the experiments in (A) using DBSCAN. Error bars, SD. D Quantification of the number of protein copies in interphase NPCs (NPC), mitotic clusters and kinetochores of the experiments in (A) using DBSCAN. Error bars, SD. E Quantification of the cluster width of interphase NPCs (NPC), mitotic clusters and kinetochores of the experiments in (A) using DBSCAN. Error bars, SD.
Figure 2(A) Colocalization analysis of KIF3A/B/KAP3 with APC by immunostaining of hippocampal primary neurons.(D) Pull-down analysis of the binding of APCARM with KIF3/KAP3.
Figure 3(D) XL-MS analysis of the interaction sites between KIF3/KAP3 and APCARM. Two crosslinked site pairs were found to correspond to the possible KIF3/KAP3-APCARM binding region marked by asterisks in C. Cartoon diagrams, sequence alignment and LC-MS data are shown with crosslinked sites highlighted.
Figure 4(A) HS-AFM imaging of KIF3/KAP3-APC particles.(B) Time lapse HS-AFM images of a representative ABK-APCARM particle with a continuous tapping force.(C) Representative HS-AFM images of ABK-APCARM illustrating the dissociation dynamic. Continuous tapping of AFM triggered the dissociation of APCARM from ABK. Red arrows indicate the binding interface between ABK and ABK-APCARM.(D) Statistical analysis of the AFM-based ABK and ABK-APCARM particle conformations. 200 frames extracted from three independent AFM movies of ABK and ABK-APCARM were used for the analysis. Distance from the coiled coil distal tip to the docking domain (length of the protrusion as shown by the dotted lines) was calculated and shown as violin plots. Data derived from each of the three movies were indicated by circles, triangles and rectangles, respectively. Based on the distal-center distance, particle conformations were classified into three forms, L (cargo-locked globular form), T (Transitional curved form) and D (cargo-docked elongated form). Unpaired t test was used to evaluate the statistical significance. The solid and dotted lines indicate the median and quartiles, respectively. The AFM images shown here are re-used in the figure 5A.
Figure 1B. Jac1488 was imported into purified mitochondria for indicated times and treated with Proteinase K (PK) or left untreated as indicated. For comparison, 47 pmol of precursor were loaded (lane 1). Prec., purified precursor protein; p, precursor; m, mature protein; ∆ψ, membrane potential.C. Jac1488 import into mitochondria as in B, right side; Jac1488 protein dilution series, left side.D. Quantification of import of Jac1488 (maximal signal at 15 min import time, 100%); error bars indicate (SEM) (n=3). E. Quantification of absolute imported amounts in picomoles Jac1488 per µg of mitochondria; error bars indicate SEM (n=3).
Figure 2A. Steady state protein levels in wild-type (WT) and Tim50-depleted mitochondria visualized by Western blotting and immunodetection with indicated antisera.B. Jac1488 was imported into wild-type (WT) and Tim50-depleted mitochondria for indicated times and samples were treated with Proteinase K. p, precursorC. Quantification of Jac1488 import into wild-type (WT) and Tim50-depleted mitochondria. The amount of imported protease-protected protein in WT mitochondria at 15 min was set to 100%; error bars indicate SEM (n=3).D. [35S]Jac1 was imported into wild-type (WT) and Tim50-depleted mitochondria for indicated times and samples were treated with Proteinase K. p, precursor; m, mature.E. Quantification of [35S]Jac1 import into wild-type (WT) and Tim50-depleted mitochondria. The amount of imported protease-protected protein in WT mitochondria at 15 min was set to 100%; error bars indicate SEM (n=3).F. Steady state protein levels in wild-type (WT) and tim44-804 mitochondria visualized by Western blotting and immunodetection with indicated antisera.
Figure 3B. Purified pAmJac1 was conjugated with different fluorophores as indicated. Detected without bleed through under fluorescence imaging conditions.C. Jac1488 and pAmJac1 conjugated to DyLight 488, 680, and 800 were imported into wild-type mitochondria for indicated times and samples were treated with Proteinase K. Prec., purified precursor protein; p, precursor; m, mature.E. Jac1488 and pImJac1 conjugated to DyLight 488, 680 and 800 were imported into wild-type mitochondria for indicated times and samples treated with Proteinase K. Prec., purified precursor protein; p, precursor; m, mature.
Figure 4D. Quantification of picomoles of pAmJac1680 imported per µg mitochondria as assessed in 96-well format; error bars indicate SEM (n=8).E. pAmJac1680 was imported into purified mitochondria and samples analyzed by SDS-PAGE or in 96-well format. The amount of imported, protease-protected samples at 15 min was set to 100% in each case; error bars indicate SEM (n=8 for 96-well plate analysis, n=3 for SDS-PAGE).G. pAmJac1680 was imported into wild-type (WT) and Tim50-depleted mitochondria for indicated times and samples treated with proteinase K. Samples were analyzed in 96-well format and by SDS-PAGE. The amount of imported protease-protected protein in WT mitochondria at 15 min was set to 100%; error bars indicate SEM (n=5 for 96-well plate analysis, n=4 for SDS-PAGE).
Figure 5A. Atp5488 (left panel) or [35S]Atp5 (right panel) were imported into mitochondria for indicated times and in the presence or absence of a membrane potential (∆ψ). After proteinase K treatment, samples were separated by SDS-Page and analyzed by fluorescence scanning or digital autoradiography. Prec., purified precursor protein; p, precursor; m, mature protein.B. Import of Atp5488 with increasing concentrations of CCCP to study dependency of import on membrane potential (∆ψ). P, precursor; m, mature protein.D. [35S]Atp5 was imported with increasing CCCP concentrations as described in B. Samples were separated by SDS-PAGE and analyzed by digital autoradiography. E. Quantification of import of [35S]Atp5 with increasing CCCP concentrations. The amount of imported protease-protected protein in the absence of CCCP was set to 100%; error bars indicate SEM (n=3).G. Atp5488 (left panel) or [35S]Atp5 (right panel) were imported into wild-type and atp21∆ mitochondria for indicated times and in the presence or absence of a membrane potential (∆ψ). After proteinase K treatment, samples were solubilized in digitonin buffer and separated by BN-PAGE. Aliquots of the samples was analyzed by SDS-PAGE (lower panels). Subsequently proteins were visualized by fluorescence scanning or digital autoradiography. Complex V dimer, (CVD) and monomer (CVM). Prec., purified precursor ; p, precursor; m, mature protein.
Figure 1(A) PolyP accumulation in vivo. The polyP content of wild-type cells was set to 100%. Knockout of Vtc1, Vtc2, Vtc3 or Vtc4 impacts cellular polyP levels. Data show the mean±s.d (biological replicates n=3).PolyP synthesis curves of purified endogenous Vtc4/Vtc3/Vtc1 and Vtc4/Vtc2/Vtc1 complexes at different ATP concentrations in the absence or presence of 10 mM InsP6 in vitro.PolyP synthesis by isolated vacuoles carrying VTC complexes in the absence or presence of 1 µM 5-InsP7 in vitro.
Figure 3Profile of the pore radius of the Vtc4/Vtc3/Vtc1 complex.
Figure 4Cellular polyP content of Vtc4p point mutants expressed under the control of their native promoters in the vtc4Δ background. Data show the mean±s.d (biological replicates n=3).Cellular polyP content of VTC4 and VTC3 point mutants expressed under the control of their native promoters in the vtc4Δ and vtc3Δ(vtc2Δ) backgrounds, respectively. Data show the mean±s.d (biological replicates n=3).Cellular polyP content of VTC4 point mutants expressed under the control of their native promoters in the vtc4Δ backgrounds. Data show the mean±s.d (biological replicates n=3).
Figure 5PolyP synthesis by isolated vacuoles carrying Vtc4(ΔSPX)/Vtc3/Vtc1 complex, Vtc4/Vtc3(ΔSPX)/Vtc1 complex or Vtc4/Vtc3/Vtc1 complex in the absence or presence of 1 μM 5-InsP7 or 1,5-InsP8 in vitro. The reaction system is detailed in Methods. Data show the mean±s.d (biological replicates n=3).PolyP synthesis by isolated vacuoles carrying Vtc4/Vtc3(R223E)/Vtc1 complex, Vtc4/Vtc3(R226E)/Vtc1 complex or Vtc4/Vtc3/Vtc1 complex in the absence or presence of 1 μM 5-InsP7 or 1,5-InsP8 in vitro. The reaction system is detailed in Methods. Data show the mean±s.d (biological replicates n=3).
Figure 6PolyP synthesis curves of purified endogenous Vtc4/Vtc3/Vtc1 and truncated Vtc4/Vtc3(Δlp)/Vtc1 complexes at different ATP concentrations in the absence or presence of 10 mM InsP6 in vitro. The reaction system is detailed in Methods. Data show the mean±s.d (biological replicates n=3). (D) PolyP synthesis by isolated vacuoles carrying Vtc4/Vtc3(CL2-Ala6)/Vtc1 complex, Vtc4/Vtc3(CL2-Asp6)/Vtc1 complex or Vtc4/Vtc3/Vtc1 complex in the absence or presence of 1 μM 5-InsP7 or 1,5-InsP8 in vitro. The reaction system is detailed in Methods. Data show the mean±s.d (biological replicates n=3).
Figure 7Cellular polyP content of VTC4, VTC3 and VTC1 point mutants expressed under the control of their native promoters in the vtc4Δ, vtc3Δ(vtc2Δ) and vtc1Δ backgrounds, respectively. Δ indicates that the entire subunit was knocked out. FL indicates full length, indicating that the subunit has not been modified in any way. Data show the mean±s.d (biological replicates n=3).
Figure 1(C) Representative images of HeLa cells transiently transfected with GST-GFP-CENP-E2605-2701 and immunostained with PRC1 and CENP-C, scale bar 10μm (n=4).(E) Top, Representative images of live HeLa cells in cytokinesis transiently transfected with GFP-CENP-E2605-2701 and mutants (green), incubated with SiR-tubulin (red). Scale bar, 10μm. Bottom, linescans showing the fluorescence intensity average and standard error of the mean (SEM) for the GFP-CENP-E2605-2701 and mutants and tubulin across the cell midbody. For GFP-CENP-E2605-2701, n is the number of cells. n=11 and for the GFP-CENP-E2605-2701 mutants FDN2661LTT, YF2660AA, FF2644AA, n= 15, 16 and 25 respectively. Biological independent replicates were respectively 2, 3, 2 and 1.
Figure 2(A) Top. Size-exclusion chromatography elution profile of MBP-CENP-E2605-2701 (green), PRC11-168 (pink) and MBP-CENP-E2605-2701/PRC11-168 (orange). Bottom, Coomassie-stained gel showing the size-exclusion chromatography profile of PRC11-168 (pink), MBP-CENP-E2605-2701 (green) and MBP-CENP-E2605-2701/PRC11-168 (orange). A shift in the elution volume was only seen in the presence of both CENP-E and PRC1. (B) Top. Size-exclusion chromatography elution profile of MBP-CENP-E2605-2701FDN (green) and MBP-CENP-E2605-2701 FDN/PRC11-168 (orange). Bottom, Coomassie-stained gel showing the size-exclusion chromatography profile of MBP-CENP-E2605-2701FDN (green) and MBP-CENP-E2605-2701 FDN/PRC11-168 (orange). No shift in the elution profile was observed.
Figure 3(B) Representative images of live HeLa cells in mitosis transiently transfected with either GFP-Kif4A1133-1165 or GFP-Kif4A1133-1232 incubated with SiR-Tubulin. Scale bar, 10μm. Quantification of cells with GFP-Kif4A1133-1165 (n=12) or GFP-Kif4A1133-1232 (n=13) localization to the central spindle. Data represented from 2 independent experiments.(C) A shift in the elution volume was only seen in the presence of both Kif4A and PRC1. Size-exclusion chromatography elution profile of GST-Kif4A1133-1232 (green) and PRC11-168 (pink), or together (orange). Bottom, Coomassie-stained gel showing the size-exclusion chromatography profile of GST-Kif4A1133-1232 (green), PRC11-168(pink) and GST-Kif4A1133-1232/PRC11-168 (orange).
Figure 4(A) Representative images of GFP-PRC1 (magenta) mixed with rhodamine-microtubules. Fire blue-green intensity LUT used to show tubulin intensity and microtubule overlaps. (B) Representative images of 647GST-CENP-E2639-2671 (yellow) mixed with rhodamine-microtubules.(D) Representative immunofluorescence images of HeLa cells stained for DNA, microtubules, PRC1 and with Alexa647-labelled GST-CENP-E2639-2671 showing it also recognizes PRC1 in cells. Experiments were repeated >3 times.(F) Representative kymograph showing microtubule-microtubule sliding in the presence of 2.5 nM PRC1 and 50 nM full-length CENP-E. (G) Example kymograph showing a free microtubule sliding until reaching the end of the immobilized microtubule where it slows down to a stall.
Figure 5(D) Live-cell imaging of metaphase spindles in HeLa cells transiently transfected wild-type, phosphomimetic and non-phosphorylatable mutants of GFP-CENP-E2605-2701 (monomeric) and GFP-GST-CENP-E2605-2701 (dimeric) and stained for tubulin using SiR-Tubulin. The fraction of cells localizing to the overlapping microtubules is represented as a percentage. Scalebar, 10 μm.(G) Bar graph showing mean and standard error for GFP fluorescence intensity at peak fluorescence for GFP-GST-CENP-E2605-2701 and mutants at 9.7μm, quantified in F. Asterisks indicate ordinary One-way Anova test significance value. ****P<0.0001.
Figure 6(B, C) Elution profile from a size-exclusion chromatography (SEC, black line, left y-axis) run with subsequent multi-angle light scattering (MALS, right y-axis) analysis for PRC11-168 and PRC11-168 MEE. Outcome of the MALS analysis for the peak is presented in pink (molecular weight, right y-axis).(D) Fire purple-yellow intensity LUT used to show tubulin intensity and microtubule overlaps. Microtubules are shown alone or incubated with 2.5 nM full-length PRC1 WT or PRC1 MEE. Scalebar, 10 μm. Experiment was replicated 2 times.
Figure 7(A) Representative immunofluorescence images of HeLa cells in late anaphase and telophase, expressing GFP-PRC1-WT or -MEE and depleted for endogenous PRC1 using a PRC1 siRNA and a PRC1 sgRNA after doxycycline-induced Cas 9 expression. Microtubules, GFP and CENP-E are in white, green and red respectively. DNA is in blue. (B) Graph showing the percentage of cells with a PRC1-localized at the site of abscission for cells treated in (A). Mean and standard deviation are presented, n= 156 and 141 for cells expressing PRC1-WT and -MEE respectively. Data from 4 biological replicates and 1 technical replicate. Asterisks indicate a T-test significance value. ****P<0.0001. (C) Graph showing the percentage of cells with CENP-E localized at the abscission site for cells treated in (A), mean and standard deviation are presented, n= 58 and 66 for cells expressing PRC1-WT and -MEE respectively. Data from 2 biological replicates. p-value (p=0.11) calculated for an unpaired T-test.(G) Representative live-cell images of HeLa cells expressing GFP-PRC1-WT or -MEE and depleted for endogenous PRC1 using a PRC1 siRNA and a PRC1 sgRNA after doxycycline-induced Cas 9 expression. Microtubules (SiR-Tubulin) and DNA (SPY650) are shown in red. The cell membrane (CellMask) is highlighted in white and GFP-PRC1 is in green. Scalebar, 10 μm.(J) Representative immunofluorescence images of cells expressing GFP-PRC1-WT or -MEE after 72 hours siRNA depletion and doxycycline-induced knockout of endogenous PRC1. DNA, microtubules and GFP-PRC1/GFP-PRC1-MEE are in blue, red and green respectively. Scalebar, 10μm.
Figure 1A-C) A) Bilateral bowing deformity and short femora and tibiae with atypical growth plates, and B) short and bowed humerus at age 4.5 years, C) vertebral fractures (arrows) on lateral imaging at age 9 years.
Figure 2(A-H) Transiliac bone sample of the patient at age of 10 years viewed in the light microscope. A-E) Goldner stained sections, F-H): Giemsa stained sections. A) Overview of the entire sample shows scarcity of mineralized bone (stained in green). Black arrowheads point towards the very thin cortices (right side, only a fractured cortex is visible). B) Trabecular features are mostly small and isolated. C) Black arrows point toward three bone resorbing osteoclasts leading to the disconnection of two trabecular features. D) Cortical bone viewed under polarized light: white arrows point towards collagen fibers arranged in a parallel concentric way around osteon next to a region where collagen fibers are randomly arranged, as typical for woven bone (white asterisk). E) Detail from cortical bone showing roundish cells on the periosteal side surrounded by a mainly red, thus, poorly mineralized matrix (white stars). F), G) Details from the cortex in Giemsa stained section show areas with unmineralized cartilage (black arrows) and mineralized woven bone and or mineralized cartilage (dark purple, black asterisks), mineralized bone is stained pink. H) Trabecular feature with mineralized cartilage inclusion stained deep purple (arrowhead), surrounded by bone tissue stained pink. Note many osteoblasts on the trabecular surface (black arrows)
Figure 3A) Family pedigree with SEC16B homozygosity in the patient and heterozygosity in the mother and one brother. B) Validation of the SEC16B mutation by Sanger sequencing. Sanger sequencing of gDNA of the patient, brother, mother and healthy control. Magnification of the electropherogram shows the position c.424 and the base change CT.
Figure 4C-F) C) COL1A1 and COL1A2 expression in the ER of SEC16B patient and control fibroblasts assessed by western blotting. KDEL, Calnexin, BIP and PDI, all ER-resident proteins were used as ER markers. E) Golgi complex was isolated according to the method described in Current Protocols in Cell Biology, Unit 3.9 from healthy control cells and patient derived fibroblasts. Subsequently, COL1A1 and COL1A2 expression in the Golgi complex of SEC16B patient and control fibroblasts were determined by western blotting. GM130 (Golgi-resident protein) was used as marker for Golgi complex. All cells were treated with ascorbic acid 0.05 mg/ml for 20 hours before the Golgi complex was isolated. D, F) Quantification of the bands that was done with Bio-Rad Image Lab 6.1 software to calculate the expression levels. COL1A1 and COL1A2 band intensities were either normalized to (D) calnexin or (F) GM130. The normalized intensities were reported as fold change patient to control cells. Columns represent the mean of three (D) or two (F) independent experiments (raw values: 0.27 and 0.53 for COL1A1 and 0.29, 0.21 for COL1A2) respectively. Biological replicates were performed; bars, standard error of the mean (SEM); *significant (p < 0.05) differences obtained by using unpaired two-tailed t test
Figure 5A) Patient and control fibroblasts were plated on glass cover slips. After 24 hours, cells were fixed and stained for SEC31 to label ERES. The scale bars are 10 µm. B) Quantification of the number of ERES per area from cells processed. The values are from four independent experiments, biological replicates. 71 cell per experiment were acquired. Bars are means ± standard error of the mean (SEM); *significant (p < 0.05) differences obtained by using a paired two-tailed t-test.C) Patient and control fibroblasts were electroporated with plasmids encoding mCherry-tagged Mannosidase II (ManII) as part of a RUSH construct. After 24 hours, cells were fixed (T0) or treated with 40 mM biotin for 20 minutes (T20) followed by fixation and immunostaining for GM130 to label the Golgi complex. The scale bars are 10 µm. D) quantification of 59 to 81 cells from three independent RUSH experiments (biological replicates). Biological replicates were performed. Central band represents the median. Whiskers are minimum and maximum values. Outliers are defined as greater than 1.5 fold of upper quartiles.** highly significant (p < 0.005) differences obtained by unpaired two tailed t-test